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Journal of Biochemistry Advance Access published online on November 6, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp182
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Purification of Paracoccidioides brasiliensis catalase P; subsequent kinetic and stability studies.

Ronney Fernandes Chagas1, Alexandre Melo Bailão1, Kátia Flavia Fernandes2, Michael S. Winters3, Maristela Pereira1 and Célia Maria de Almeida Soares1,*

1Laboratário de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, Goiás, Brazil; 2Laboratório de Química de Proteínas, Universidade Federal de Goiás; 3 University of Cincinnati, College of Medicine, Cincinnati, Ohio, USA.

*Correspondence author: Célia Maria de Almeida Soares, Laboratório de Biologia Molecular, ICBII, Campus II Universidade Federal de Goiás, 74001-970, Goiânia-Goiás, Brazil. Tel./fax: +55 62 35211110; e-mail: celia{at}icb.ufg.br

Received September 22, 2009; Accepted October 14, 2009


  Abstract

Catalases are essential components of the cellular equipment to cope with oxidative stress. Here we have purified a highly abundant catalase P of Paracoccidioides brasiliensis (PbCatP) that is preferentially expressed in the parasitic yeast phase. This oxidative stress-induced protein was isolated from yeast cells grown in the presence of 15 mM of hydrogen peroxide. We have used consecutive steps of protein precipitation and gel filtration chromatography to achieve the purified protein. Protein purification was validated using matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and bioinformatics analysis. The purified enzyme showed strong similarity to small-subunit catalases. Like most monofunctional catalases, PbCatP is a homotetramer, resistant to inactivation by acidic conditions, temperature, and denaturants. Furthermore, the kinetic behavior of catalase P was observed to be different at low compared to high H2O2 concentrations. The results demonstrated that a purified catalase P of P. brasiliensis is a homotetrameric enzyme, classified as a small subunit catalase.

Key Words: Catalase P, homotetrameric enzyme, Paracoccidioides brasiliensis, protein purification and characterization, mass spectrometry


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